User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/02/10

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Proteinase K Reaction Analysis

Objectives

  1. In situ 100 nM Proteinase K reaction
  2. In vitro reaction to generate samples for Bradford analysis and gel electrophoresis
  3. Generate a Bradford calibration curve

Protocol

In situ reaction:

0.0075 mL Proteinase K stock was added to 2.9925 mL Tris/NaCl buffer for a final enzyme concentration of 100 nM. Fibers were spun down at 300 RPM for 5 minutes, and liquid extracted; the fibers were resuspended and added to the cuvette.

The Ocean Optics was set to scan every 30 seconds for 2 hours, and maintained at 37°C and stirred at 1000 RPM.

Proteinase K reaction for analysis

Monika has values for the samples prepped. Samples were run at 37°C for 2.5 hours. 300 µL of sample was extracted every 30 minutes

Bradford Calibration Curve

Monika has the data for the calibration curve samples. A 1 in 4 Bradford solution was prepared with Tris/NaCl buffer, and a calibration curve was generated.

Bradford Calibration Curve Feb10-NEW.png

Results

100nM ProtK AbsvsTime Chart.png

Absorbance vs Time for the Proteinase K In situ reaction.

100nM ProtK Kinetics Chart.png

Kinetics at 517.7 nm. Notice the plateau between 90 minutes and 120 minutes. This is seen in the absorbance vs time chart as well.